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G3139 is an antisense oligonucleotide (ODN) that can down-regulate bcl-2, thus potentially acting as a potent anticancer drug. However, effective therapy requires efficient ODN delivery, which may be achieved by employing G3139 lipoplexes. Yet, lipofection is a complex, multifactorial process that is still poorly understood. In order to shed more light on this issue, we prepared 18 different G3139 lipoplex formulations and compared them in terms of their capability to transfect MCF-7 breast cancer cells. Each formulation was composed of a cationic lipid and sometimes a helper lipid. The cationic lipid was either DOTAP (N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride), DC-CHOL (3ss[N-(N',N'-dimethylaminoethane)carbamoyl]-cholesterol), or CCS (ceramide carbomoyl spermine). The helper lipid was either DOPC, DOPE, or cholesterol. Each lipid combination existed in two different structural forms--either large unilamellar vesicles (approximately 100 nm LUV) or unsized heterolamellar vesicles (UHV). Cell proliferation assays were used to evaluate the cytotoxicity of G3139 lipoplexes, control cationic lipid assemblies, and free G3139. Western blots were used to confirm the specific activity of G3139 as an anti-bcl-2 antisense agent. We determined that treatment of MCF-7 cells with G3139:CCS lipoplexes (UHV-derived) produced a maximal 50-fold improvement in antisense efficacy compared to treatment with free G3139. The other G3139 lipoplexes were not superior to free G3139. Thus, successful lipofection requires precise optimization of lipoplex lipid composition, structure, and concentration.

We have found that the profiles of total phospholipids in malignant breast cancer cell lines change going from hormone sensitive to highly hormone resistant cells lines. In particular, two phospholipid components that were absent or at very low levels in hormone sensitive MCF7 cells and moderately hormone sensitive cell lines (MIII, LCC2) were found in relatively high proportions in highly hormone resistant cell lines (MB435, MB231). These two components were shown to be the alkylacylphosphatidylcholine (AAPtdC) and the unsaturated analog plasmenylphosphatidylethanolamine (plasmenyl-PtdE). Another component phosphatidylethanolamine (PtdE) increased in correlation with the degree of hormone insensitivity. This was shown using 31P NMR spectroscopy of lipid extracts of the cells, and was confirmed using HPLC analysis, as well as other techniques. The significance of these results for the metabolic characteristics of these cell lines is related to the therapeutic responsiveness of breast cancer.

Objective: To assess our long-term complications from complete axillary lymph node dissection (AXLND) in patients with breast cancer., Summary Background Data: Complete AXLND as part of the surgical therapy for breast cancer has come under increased scrutiny due the use of the sentinel lymph node (SLN) biopsy technique to assess the status of the axillary nodes. As the enthusiasm for the SLN technique has increased, our impression has been that the perceived complication rate from AXLND has increased dramatically while the negative aspects of the SLN technique have been underemphasized., Methods: Female patients seen in routine follow-up over a 1-year period were eligible for our retrospective study of the long-term complications from AXLND if they were a minimum of 1 year out from all primary therapy; ie, surgery, radiation, and/or chemotherapy. All patients had previously undergone either a modified radical mastectomy (MRM) or a segmental mastectomy with axillary dissection and postoperative radiation (SegAx/XRT). All patients had a Level I-III dissection. Objective measurements, including upper and lower arm circumferences and body mass index (BMI), were obtained, and a subjective evaluation from the patients was conducted., Results: Ninety-four patients were eligible for our study; 44 had undergone MRM, and 50 had undergone SegAx/XRT. The average number of nodes removed was 25.6 (standard deviation, 8). Thirty-three percent of the patients had positive nodal disease, 95% of the patients had an upper arm circumference within 2 cm of the unaffected side, and 93.3% had a lower arm circumference within 2 cm of the unaffected side. Subjectively, 90.4% of the patients had either no or minimal arm swelling, and 96.8% of the patients had "good" or "excellent" overall arm function. The most common long-term symptom was numbness involving the upper, inner aspect of the affected arm (25.5%)., Conclusions: Our data indicate that a complete AXLND can be performed with minimal long-term morbidity. The lower the morbidity of AXLND, the less acceptable are the unique complications of the SLN technique.

Magnetic resonance spectroscopy (MRS) methods have provided valuable information on cancer cell metabolism. In this study, we characterized the 31P-MR spectra of breast cancer cell lines exhibiting differences in hormonal response, estrogen receptors (positive/negative), and metastatic potential. A correlation was made between the cytotoxic effect of antimitotic drugs and changes in cell metabolism pattern. Because most anticancer drugs are more effective on proliferating cells, our study attempted to elucidate the metabolic profile and specific metabolic changes associated with the effect of anticancer drugs on proliferating breast cancer cell lines. Accordingly, for the 31P-MRS experiments, cells were embedded in Matrigel to preserve their proliferation profile and ability to absorb drugs. The MRS studies of untreated cells indicated that the levels of phosphodiesters and uridine diphosphosugar metabolites were significantly higher in estrogen receptor-positive and low metastatic potential cell lines. 31P-MRS observations revealed a correlation between the mode of action of anticancer drugs and the observed changes in cell metabolic profiles. When cells were treated with antimicrotubule drugs (paclitaxel, vincristine, colchicine, nocodazole), but not with methotrexate and doxorubicin, a profound elevation of intracellular glycerophosphorylcholine (GPC) was recorded that was not associated with changes in phospholipid composition of cell membrane. Remarkably, the rate of elevation of intracellular GPC was much faster in cell population synchronized at G2-M compared with the unsynchronized cells. The steady-state level of GPC for paclitaxel-treated cells was reached after approximately 4 h for synchronized cells and after approximately 24 h (approximate duration of one cell cycle) for the unsynchronized ones. These observations may indicate a correlation between microtubule status and cellular phospholipid metabolism. This study demonstrates that 31P-MRS may have diagnostic value for treatment decisions of breast cancer and reveals new aspects of the mechanism of action of antimicrotubule drugs.

We have used diffusion-weighted proton magnetic resonance spectroscopy (DWMRS) to noninvasively selectively observe only the intracellular metabolites of breast cancer and melanoma cell lines in vitro in real time. Breast cancer cell lines representing different stages in breast cancer progression were chosen for study. Intracellular biochemical profiles of six cell lines perfused in alginate beads were obtained. Spectral differences between groups of cell lines, including choline, lactate, and threonine peaks, were investigated. We also monitored response to the antineoplastic agent, lonidamine (LND), as a function of time and drug concentration in perfused cancer cells. Previous studies reported that this drug induced intracellular acidification and lactate accumulation. Diffusion weighted proton spectra demonstrated a 2- to 9-fold increase in the intracellular lactate signal as a response to LND treatment in several cancer cell lines. These results are consistent with the hypothesis that the principal mechanism of LND in some cancer cells is marked inhibition of lactate transport. Moreover, we have shown that there is a factor of two to three between the response of melanoma cells and that of some types of breast cancer cells. The higher sensitivity of the melanoma cells, as predicted by proton DWMRS, was correlated with changes in water-suppressed magnetic resonance spectra and confirmed by a biological assay. This study demonstrates the feasibility of using DWMRS for monitoring intracellular metabolism and for studying the effects and mechanisms of action of anticancer drugs. We believe that this method can be used for noninvasive clinical applications, such as the differentiation between benign and malignant tissue, real-time monitoring of response to therapy, dose response, and toxicity effects.

Tumor necrosis factor-alpha inhibited growth of cultured MCF-7 human breast cancer cells in a dose dependent manner. Tumor necrosis factor-alpha also markedly increased glucose consumption, and its cytotoxicity was modified by glucose concentrations in the growth medium; higher glucose levels were associated with increased cell survival. However, when the cells were perfused in physiological conditions, very high levels of tumor necrosis factor-alpha (200 ng/ml) in the perfusion solution had no inhibitory effects. Moreover, tumor necrosis factor-alpha had no effects on 31P nuclear magnetic resonance spectra of the perfused cells. In the traditional growth inhibition assays, cells are incubated for several days with a drug, a situation where their metabolism is altered due to the depletion of nutrients, the accumulation of toxic waste materials and pH changes. Perfusion experiments are more relevant to in vivo conditions, and may be used for studying metabolic processes and the mechanisms of action of therapeutic agents.

Many breast tumors appear to progress from estrogen-dependent growth to a more malignant phenotype characterized by estrogen-independent growth, antiestrogen resistance, and a high metastatic potential. Utilizing 31P NMR spectroscopy on human breast cancer cells growing in vitro, we have investigated the effects of 17 beta-estradiol and tamoxifen on the metabolic/bioenergetic spectra of a series of human breast cancer cells that vary in their estrogen and antiestrogen responsiveness. A comparison of baseline spectra associates higher levels of phosphodiesters and UDP-glucosides (e.g. UDP-glucose, UDP-N-acetylglucosamine), and lower phosphocholine/glycerylphosphocholine and phosphocholine/phosphoethanolamine ratios, with the acquisition of estrogen-independent growth in estrogen receptor expressing cells. No metabolic changes are clearly associated with the metastatic phenotype. Whilst estrogen treatment produces no consistently significant spectral changes in any of the cell lines, the estrogen-independent and estrogen-responsive MCF7/MIII cell line responds to tamoxifen treatment by significantly increasing all spectral resonances 30%-40% above baseline values. This may reflect a tamoxifen-induced change to a more differentiated or apoptotic phenotype, or an attempt by the cells to reverse the inhibitory effects of the drug. The ability to detect metabolic changes in response to tamoxifen by NMR spectroscopy may provide a novel means to identify those tumors that are responsive to antiestrogen therapy.

The basis for the use of nuclear magnetic resonance (NMR) spectroscopy as a tool to study the metabolism of breast cancer cells is described. The differences between proton (1H), carbon (13C), and phosphorus (31P) NMR methods is explained, and the techniques of cell extracts, cell suspensions and perfusion methods for cells are detailed. In order to perfuse cells they are preferably trapped in a gel matrix, either in the form of a thread or a bead. The gel must have appropriate properties that enables efficient oxygenation and availability of nutrients and drugs. The metabolic effects of perfusion of breast cancer cells with nutrients, drugs, and hormones are reported, and the clinical relevance of these results and methods are outlined.

Previously we reported that Mn(III)tetra(4-sulfonatophenyl) porphyrin, MnTPPS4, is a contrast agent which can effectively enhance tumor detection by MRI. By imaging 30 additional athymic nude mice bearing subcutaneous MCF-7 WT human breast carcinoma xenografts, we have extended dose-contrast relationships over a wide range of intraperitoneal (IP) doses ranging from 0.025 to 0.50 mmol/kg. The benefits of IP injection are higher possible doses on a volume basis and a reduction in toxicity versus IV administration. Full coronal cross-section images have been obtained on a 2-T spectrometer. Although individual tumor masses displayed different distribution patterns, reflective of their internal morphology, single doses of 0.10 mmol/kg or greater were necessary to produce a detectable effect. At a dose of 0.50 mmol/kg, marked enhancement was produced. Multiple small dosages administered over the course of several days before imaging did not produce increased enhancement. Preliminary results on the new porphyrin derivative, MnTPPS3, indicate that the ratio of the toxic dose to the effective dose may be adjustable to render this class of agents clinically useful.

The action of gossypol, a polyphenolic bisnaphthalene aldehyde, on a number of drug-sensitive and multidrug-resistant cell lines, in particular MCF-7 WT and MCF-7 ADR cells, was studied and compared to the effects of rhodamine 123. 31P nuclear magnetic resonance spectra of cells exposed to low concentrations of gossypol exhibited decreased levels of ATP, markedly increased levels of pyridine nucleotides, and decreased levels of glycerylphosphocholine. The latter effect may be related to the membrane viscosity-increasing effect of gossypol, whereas changes in the levels of pyridine nucleotides are probably due to an interference with NAD- and NADP-dependent enzymes. The effect of gossypol represents a rare example of selective and differentiated changes observed in 31P nuclear magnetic resonance spectra of cells following exposure to a drug; the effect was markedly different from that of rhodamine 123, which caused ATP depletion but no changes in the levels of glycerylphosphocholine or pyridine nucleotides. Also, the effects of gossypol and rhodamine 123 on glucose metabolism in the MCF-7 WT cells were different. Thus although both drugs caused a marked elevation of glucose uptake, an increase in lactate production exceeding that of glucose consumption, indicating an inhibition of oxidative phosphorylation, was observed only in the case of rhodamine 123. Significantly, multidrug-resistant cells exhibited strong cross-resistance to rhodamine but practically no resistance to gossypol, which emphasizes the attractiveness of the latter as a potential anticancer drug. The resistance to rhodamine 123 and sensitivity to gossypol was also observed with cells transfected with the MDR1 gene, showing that the difference in toxicity is mainly due to the different response to the P-170 drug efflux pump.

The glycolytic inhibitor 2-deoxyglucose (2-DG) was tested as a potential chemotherapeutic agent for drug-resistant cancer cells. Previously it was found that Adriamycin-resistant human MCF-7 breast cancer cells (ADR) exhibit an enhanced rate of glycolysis compared to their parent wild-type (WT) cell line (R. C. Lyon et al., Cancer Res., 48: 870-877, 1987). We now describe a specific toxic effect of 2-DG on the ADR cells, which is more than 15-fold greater than for WT cells. Using 31P magnetic resonance spectroscopy of perfused MCF7 cells we continuously monitored the accumulation of 2-deoxyglucose 6-phosphate together with concomitant changes in other phosphate-containing metabolites. Kinetic measurements demonstrated that ADR cells accumulated 2-deoxyglucose 6-phosphate faster and to a greater extent than WT cells, while their depletion of high energy compounds (ATP, phosphocreatine) was more pronounced and became irreversible earlier. The phosphorylation of 2-DG could be followed more effectively by the use of 13C magnetic resonance spectroscopy of 2-DG enriched with 13C at C-6, since the signals of 2-DG and 2-deoxyglucose 6-phosphate are clearly resolved and, unlike 31P magnetic resonance spectroscopy, there are no other interfering signals. With the use of this technique with ADR and WT cells the rate of phosphorylation of 2-DG was found to be 11.2 x 10(-4) and 6.5 x 10(-4) mmol/min/mg protein, respectively. The results of these studies indicate that differences in the biochemistry of energy metabolism of resistant cells may make them targets for energy antimetabolites.

Using 31P nuclear magnetic resonance spectroscopy we have noninvasively observed metabolic control through the cytidine pathways of phosphatidylcholine and phosphatidylethanolamine synthesis in intact actively metabolizing MDA-MB-231 human breast cancer cells. Perfusion with the phospholipid precursors ethanolamine or choline (2 mM) indicates that the cytidylyltransferase enzymes are rate limiting for both pathways. Complete inhibition of choline kinase with ethanolamine allowed the observation of the utilization of phosphocholine by the rate-limiting enzyme choline-phosphate cytidylyltransferase. The rate was dependent on the phosphocholine concentration. Inhibition of glycerophosphorylcholine phosphodiesterase with accumulation of substrate was also observed and allows an estimate of the flux through the degradative pathways. The human lymphoma cell line MOLT-4 was also found to contain high levels of phosphocholine and phosphoethanolamine. The levels of these precursors in the MOLT-4 line are lowered by 40% after 6 h when perfused with high dose 1-beta-D-arabinofuranosylcytosine (Ara-C) (400 microns) but are unaffected by 2 microns Ara-C or dideoxycytidine. High dose Ara-C also resulted in lysis in 8-10 h. However, the MDA-MB-231 cell line which is not sensitive to Ara-C showed no change in its spectrum when perfused with Ara-C. A potential mechanism based on classic phospholipid metabolism for the lytic effect of high dose Ara-C is discussed.

Recently, Vasanthakumar and Ahmed reported (Vasanthakumar, G.; Ahmed, N.K., Cancer Communications 1:225-232; 1989) a complete inhibition of the multiple drug resistance gene (MDR1) in the K562/III erythroleukemia cells, using a 15 bases-long methylphosphonate oligodeoxynucleotide analog. The sequence used, however, contained three mismatches relative to the corresponding fragment of the human MDR1 gene and, hence, the results reported cannot at present be regarded as a classical antisense effect. We have made attempts to inhibit the expression of the MDR1 gene in MCF-7 human breast cancer cells selected for resistance to Adriamycin using phosphorothioate analogs of oligodeoxynucleotides. Studies with model 35S-labeled-phosphorothioates indicated poor uptake of the compounds into the cells; the radioactivity was located mainly in the soluble fraction (cytoplasm), but membranes and the nuclear fraction were also labeled. Unmodified oligodeoxynucleotides were toxic to the cells, whereas the phosphorothioates were not. The MDR1 inhibition with phosphorothioates was studied by measuring their effects on adriamycin toxicity and by immunocytochemical titration of P170. Elevation of adriamycin cytotoxicity consistent with a decreased drug resistance was observed with one antisense sequence, but the immunocytochemical assay indicated only slight inhibition of the synthesis of P170. In the wild type (drug sensitive) MCF-7 cells phosphorothioates decreased adriamycin toxicity in a sequence-independent manner. The results indicate that the effects of antisense oligodeoxynucleotides on cells are complex. Computer simulation of the secondary structure of MDR1 mRNA indicated not only extensive folding but, also, the presence of many regions not involved in intramolecular hybridization, which are of potential interest as targets for antisense oligodeoxynucleotides.

The criteria for tumor-specific MRI contrast agents are considered. The metalloporphyrins have been shown to be potential tumor-specific contrast agents due to their selective retention by cancer cells. The Mn(III) water-soluble porphyrin complexes are preferred on the basis of relaxivity and stability. Protein binding in human plasma enhances the relaxivity of Mn(III)-tetraphenylsulfonato-porphyrin (MnTTPS). In preliminary work this agent was shown to enhance MRI contrast at 0.5 T in subcutaneous human colon carcinomas grown in nude mice. Here we report further evidence of enhanced contrast in the same system, and extend this work to human breast tumors in nude mice using a 2 T animal imager. Questions of metalloporphyrin stability, toxicity and dose-contrast relationship are discussed.

We describe the use of 2-deoxy-D-[6-13C]glucose to follow simultaneously, by 13C NMR, both transport and phosphorylation to its 6-phosphate form, in MCF-7 breast cancer cells in vitro and in vivo in subcutaneous tumors in nude mice.

31P magnetic resonance spectra of perfused human breast cancer cells with the phenotype of pleiotropic drug resistance exhibit striking differences in the levels of phosphate metabolites from the wild-type, drug-sensitive parent cell line. Resistant cells demonstrated elevated levels of phosphocreatine and depressed levels of phosphomonoesters, phosphodiesters, and diphosphodiesters. These differences may reflect significant alterations in the control of bioenergetic metabolism between drug-resistant and -sensitive cells.

Glucose utilization and lactate production have been monitored as a function of time using 13C magnetic resonance spectroscopy and [13C1]-glucose with perfused wild type MCF-7 human breast cancer cells and a drug-resistant (AdrR) cell line derived from them. Compared to wild type cells, AdrR cells exhibited an enhanced (3-fold) rate of glycolysis, indicating an increased demand for ATP production. We have investigated the effects of glucose depletion and azide, an inhibitor of oxidative phosphorylation, on the levels of intracellular phosphates (Pi, ATP) and intracellular pH using 31P magnetic resonance spectroscopy and on the rates of glycolysis. In both cell lines, ATP levels and the rates of glucose utilization and lactate production were invariant in the presence of azide. ATP production, especially in AdrR cells, was highly dependent on active glucose metabolism. The results of these direct measurements confirm that these cells survive by predominantly utilizing glycolysis. Glutamate and myo-inositol were observed in 13C spectra of acid extracts of AdrR but not wild type cells. Both metabolites are potential substrates in drug detoxification. These differences in rates of glycolysis, ATP production, and the production of certain metabolites may reflect metabolic adaptations associated with the development of drug resistance.

31P magnetic resonance spectroscopy (MRS) has been established as an excellent noninvasive procedure for studying metabolism in cancer cells. For a full understanding of the results of in vivo MRS studies of metabolism, it is necessary to be able to conduct controlled experiments on the same intact functioning cells in a perfusion apparatus. The specific techniques used for this purpose are described, and the applications to cancer cell bioenergetics and phospholipid metabolism in drug-resistant and drug-sensitive breast cancer cells are illustrated. Spectra of the same cell line grown in a "model tumor" in natural basement membrane gel and in nude mice in vivo are compared. The results indicate a potential for the use of 31P MRS in a clinical setting for monitoring therapy and for diagnosis.

Comparative studies of the toxicity, stability, and retention of the water-soluble porphyrin, tetraphenylporphyrin sulfonate (TPPS), and its complex with Mn(III), have been made with the human breast cancer cell line MCF-7 wild type, and an adriamycin-resistant line derived from it, termed AdrR. Based on growth inhibition, we determined the maximum non-toxic concentration of MnTPPS tolerated by these cells. The integrity of MnTPPS in vitro was investigated by fluorescence microscopy, and we found that there is very little dissociation of MnTPPS within these cells within 4 days. We report novel proton magnetic resonance relaxation measurements of the bulk water of cells in a gel matrix undergoing perfusion. A slightly greater net uptake of MnTPPS in the wild-type cells was observed compared to AdrR; however, there was no significant difference in retention of MnTPPS. These results indicate that over a period of several hours the mechanism of selective retention of these compounds in tumour cells is not due to specific interaction with heme-binding protein, of which there is enhanced expression in the resistant cells. The fact that the net rate of washout of MnTPPS is approximately the same as the net rate of uptake also appears to eliminate compartmentalization or enzymatic modification of MnTPPS within these cells.